Total RNA was extracted from snap-frozen sample pellets (Ambion mirVana Kit, Life Technologies, Inc.) according to the manufacturer’s protocol. RNA quantity (QubitTM RNA BR Assay Kits, Life Technologies, Inc.) and quality (RNA6000 Nano Kit and Bioanalyzer 2100, Agilent) was determined to be optimal for each sample prior to further processing. 200 ng RNA per sample was amplified using the Total PrepTM RNA Amplification Kit (Illumina, Inc.) according to manufacturer’s protocol and quantified as above. 750 ng labeled RNA/sample was hybridized to HT-12v3 Expression BeadChips (Illumina, Inc.), scanned with an iScan (Illumina, Inc.). In GenomeStudio, probes were filtered for those detected at p<0.01 in at least 1 sample and exported for normalization in R using robust spline normalization (RSN). Hierarchical clustering was performed using Cluster, using Euclidian distance and complete linkage.
